Larval sampling at Point Whitney 10 October 2024
Today I sampled larvae (day 3 post fertilization) at Point Whitney.
Overview
Monday we conducted a strip spawn of oyster broodstock and fertilized gametes from parents with PolyIC exposure and a control group to test cross-generational priming effects.
This post details larval sampling and moving larvae to 1000 L conical tanks.
Here is a description of the spawning protocols used for this work.
Today I also exposed the 10K seed to PolyIC again as done on 7 October 2024 and took probe measurements in all tanks.
Probe measurements
I took probe measurements today from larval tanks and seed tanks and recorded measurements for larvae. Here are all the measurements:
| tank | date | time | temp.C | pH.nbs | sal.psu | initials | notes |
|---|---|---|---|---|---|---|---|
| polyIC | 10/10/24 | 08:40 | 15.4 | 7.77 | 30.23 | ash | 10K seed |
| outdoor_tray | 10/10/24 | 08:45 | 12 | 7.7 | 30.64 | ash | top |
| outdoor_tray | 10/10/24 | 08:45 | 12 | 7.66 | 30.62 | ash | middle |
| outdoor_tray | 10/10/24 | 08:45 | 11.9 | 7.65 | 30.63 | ash | bottom |
| upweller | 10/10/24 | 08:45 | 13.4 | 7.91 | 30.45 | ash | broodstock |
| upweller | 10/10/24 | 08:45 | 13.2 | 7.91 | 30.62 | ash | LCO and efforts |
| dock | 10/10/24 | 08:45 | 13.5 | 7.86 | 30.56 | ash | seed |
| 5K_1 | 10/7/24 | 12:00 | 25.6 | 8.25 | NA | carrie | PolyIC-larvae |
| 5K_2 | 10/7/24 | 12:00 | 25.9 | 8.27 | NA | carrie | PolyIC-larvae |
| 5K_1 | 10/8/24 | 09:00 | 22.9 | 8.28 | NA | carrie | PolyIC-larvae |
| 5K_2 | 10/8/24 | 09:00 | 23.6 | 8.2 | NA | carrie | PolyIC-larvae |
| 5K_1 | 10/8/24 | 16:00 | 26.7 | NA | NA | carrie | PolyIC-larvae |
| 5K_2 | 10/8/24 | 16:00 | 27.2 | NA | NA | carrie | PolyIC-larvae |
| 5K_1 | 10/9/24 | 16:00 | 27.4 | 8.29 | NA | carrie | PolyIC-larvae |
| 5K_2 | 10/9/24 | 16:00 | 28.2 | 8.32 | NA | carrie | PolyIC-larvae |
| 5K_1 | 10/9/24 | 09:00 | 23.6 | 8.4 | NA | ash | PolyIC-larvae |
| 5K_2 | 10/9/24 | 09:00 | 25.4 | 8.42 | NA | ash | PolyIC-larvae |
| 5K_1 | 10/10/24 | 08:30 | 23.2 | 8.07 | 30.9 | ash | PolyIC-larvae |
| 5K_2 | 10/10/24 | 08:30 | 23.2 | 8.06 | 30.97 | ash | PolyIC-larvae |
| 1K_5 | 10/10/24 | 10:30 | 30.5 | 7.85 | 30.89 | ash | PolyIC-larvae |
| 1K_6 | 10/10/24 | 10:30 | 30.7 | 7.84 | 30.92 | ash | PolyIC-larvae |
| 1K_7 | 10/10/24 | 10:30 | 29.9 | 7.81 | 30.99 | ash | PolyIC-larvae |
| 1K_8 | 10/10/24 | 10:30 | 29.9 | 7.81 | 31 | ash | PolyIC-larvae |
| 1K_9 | 10/10/24 | 10:30 | 29.5 | 7.85 | 30.84 | ash | PolyIC-larvae |
Larval sampling and rearing
Rearing days 1-3
Over the last few days, the PW facility folks have reared the larvae in the 5K L tanks. Tank 1 is the control group and tank 2 is the treatment group.
Conditions over the last couple of days were consistent between treatments, with temperature on 10/7 at 25.6-25.9°C and 22.9-22.6°C on 10/8. pH was 8.2-8.28. Flow was at 7 L per min with fluorometry measurements at 5 on 10/8. EDTA was also added on 10/8.
The diet has been pav and CC. Each tank received 10L and 14L respectively on 10/8.
On 10/9, banjos were increased from 20 µM to 40 µM. Fluoro measurements were brought up to 10 in the afternoon. On 10/9, temps were 23.6°C in the control Tank 1 and 25.4°C in the treated Tank 2. Fluoro measurements were 12-14 in each tank and pH was 8.2-8.4. Diet remained the same with 6L pav added and 9L new nano dilute in each tank. Soda ash added on 10/9. EDTA also added.
Dropping day 3
Today we dropped the tanks onto a 40 uM seive. After the tanks were all drained onto the seive, we had concentrated larvae. Here are some pictures of that process.
Here is what the larvae looked like from the control group:
And here is the seive from the treated group:
Sampling day 3
I sampled n=15 larval samples from each of the 5K tanks to equal n=15 samples per treatment group. I did this by gently scraping a small sample off the filter and adding to tubes with 300 uL RNA/DNA shield. I scooped a sample with a small spatula and cleaned the spatula with 10% bleach and DI water between samples.
Tubes 1-15: Control
Tubes 16-30: Treated
I took the samples home to be frozen at -20°C and will sample more next week.
Next week I will sample n=6 per replicate conical tank (see details below on conical tanks).
Moving to 1K L tanks on day 3
Carrie then concentrated the larvae and counted them and we allocated them into 1,000L tanks. We only kept larvae that were retained on a 60 uM screen. There were very few “runts” on the lower 40 uM screen, so they were discarded.
We ended up with 54 million larvae in the control group and 29 million from the treated group.
We decided to keep density constant around the optimal level (13 million per 1K L is optimal) and divide into as many tanks per group as possible.
We ended up adding 18 million per tank in each of 3 1,000L conical tanks of the control group: Conicals 5, 6, and 7.
We added 14.5 million larvae per tank in each of 2 1,000L conical tanks of the treated group: Conicals 8 and 9.
Density is more important to keep consistent than replicate tanks. This still provides us with a duplicate batch of treated larvae.
We also added in 1g EDTA to each tank dissolved in fresh water (100 mL of water with 1 g EDTA per tank).
Tanks were fitted with a 60 uM banjo filter each. They will continue to get the same nano pav and CC diet at 10-15 fluoro levels for the next few days. They will be increased to 20-30 after day 8. Flow is the same for each tank.
10K seed
I exposed the 10K seed to PolyIC using the same solution reused from 10/7. They were expoed from 08:30-10:30.
Cleaning
I also cleaned up all of our equipment and reorganized the upstairs area. All heaters and pumps are now upstairs.
Next steps
I’ll repeat this process again on Tuesday of next week (day 9 post fertilization).
Notebook images
Here is an image of Carrie’s notebook. I’ll take pictures of this notebook again next week to see the conditions of the larvae over the next few days.
Here are the notes I took today.
